The caspase family of about 12 cysteine proteases is central to the complex intracellular process of apoptosis, or programmed cell death (Thornberry, Science (1998) 281:1312-16). Caspase activation accompanies the onset of apoptosis which has been implicated in many of the major illnesses without cures. Apoptosis is characterized by a set of morphological and biochemical changes that dying cells undergo, including condensation, shrinkage, margination of chromatin, cytoplasmic vacuolization, increased density, and fragmented nucleic with dispersal of nuclear DNA. Caspase-3 has been shown to control both cytoplasmic and nuclear events associated with Fas-mediated apoptosis in vivo (Zheng, (1998) Proc. Natl. Acad. Sci USA 95:618-23). DNase is activated by caspase in apoptotic cells (Enari, Nature (1998) 391:43-50). Caspase enzymes are good targets for small molecule inhibitors. Caspase inhibitors are potential therapeutics for certain diseases such as Alzheimers, multiple sclerosis and other neurodegenerative disorders. However, inhibiting caspases may have accompanying effect of inducing cancers and other cell proliferation effects. It is hoped that understanding the mechanisms of inducing apoptosis and inhibiting caspase activation and activity will yield drugs, e.g. to reduce damage from stroke or myocardial infarction, or prevent and treat cancer.
Peptides which include the amino acid sequence cleaved by caspases are useful probes for assaying caspase activity and thus the onset and progress of apoptosis. (Xanthoudakis, WO 00/73437; Komoriyama WO 96/13607). One critical feature of probe design is to facilitate mammalian cell membrane permeability. For high throughput drug screening it is desirable to mimic the natural state of the living cell. Therefore, the probe should enter the living cell without hypotonic shock, microinjection or other invasive or damaging techniques. For cell based high-throughput screening (HTS), another desirable property is cellular retention of the reporter dye.
Fluorescence based detection methods are important to elucidate intracellular events such as apoptosis. Enzyme substrates labelled with fluorescent dyes have been used to measure protease cleavage activity (Coyler, WO 00/50635; Weber, WO 99/18856). Fluorescent enzymatic assays have been conducted on cell-based screening systems (Dunlay, U.S. Pat. No. 5,989,835; Schroeder, U.S. Pat. No. 5,355,215) in microtiter plate, high throughput formats (Manian, U.S. Pat. No. 6,130,745; Harootunian, U.S. Pat. No. 5,589,351; Heffelfinger, U.S. Pat. No. 5,784,152; Taylor, U.S. Pat. No. 6,103,479) and digitized imaging data (Baer, U.S. Pat. No. 5,547,849
Dyes which are excited and emit fluorescence at longer wavelengths are important as labels for many molecular biology experiments (Mao, U.S. Pat. No. 6,130,101; Glazer, U.S. Pat. No. 5,565,554; Waggoner, U.S. Pat. No. 5,268,486).